Proteases from gram positive organisms

ABSTRACT

The present invention relates to the identification of a novel metalloprotease in gram positive microorganisms. The present invention provides the nucleic acid and amino acid sequences for the metalloprotease. The present invention also provides host cells having a mutation or deletion of part or all of the gene encoding the metalloprotease. The present invention provides host cells which further comprises a nucleic acid encoding desired heterologous proteins such as enzymes. The present invention also provides cleaning compositions, animal feeds and compositions used to treat a textile that include the metalloprotease of the present invention.

FIELD OF THE INVENTION

[0001] The present invention relates to metalloproteases derived fromgram positive microorganisms. The present invention provides nucleicacid and amino acid sequences of a metalloproteases identified inBacillus. The present invention also provides methods for the productionof the metalloprotease in host cells as well as the production ofheterologous proteins in a host cell having a mutation or deletion ofpart or all of the metalloprotease of the present invention.

BACKGROUND OF THE INVENTION

[0002] Gram positive microorganisms, such as members of the groupBacillus, have been used for large-scale industrial fermentation due, inpart, to their ability to secrete their fermentation products into theculture media. In gram positive bacteria, secreted proteins are exportedacross a cell membrane and a cell wall, and then are subsequentlyreleased into the external media usually maintaining their nativeconformation.

[0003] Various gram positive microorganisms are known to secreteextracellular and/or intracellular proteases at some stage in their lifecycles. Some of these proteases are produced in large quantities forindustrial purposes. However, a negative aspect of the presence ofproteases in gram positive organisms is their contribution to theoverall degradation of secreted heterologous or foreign proteins.

[0004] The classification of proteases found in microorganisms is basedon their catalytic mechanism which results in four groups: serineproteases, metalloproteases, cysteine proteases, and aspartic proteases.These categories can be distinguished by their sensitivity to variousinhibitors. For example, serine proteases are inhibited byphenylmethylsulfonylfluoride (PMSF) and diisopropylfluorophosphate(DIFP); metalloproteases by chelating agents; cysteine proteases byiodoacetamide and heavy metals and aspartic proteases by pepstatin.Further, in general, serine proteases have alkaline pH optima,metalloproteases are optimally active around neutrality, and cysteineand aspartic proteases have acidic pH optima (Biotechnology Handbooks,Bacillus. Vol. 2, edited by Harwood, 1989, Plenum Press, New York).

[0005] Metalloproteases are the most diverse of the catalytic types ofproteases. About half of the families comprise enzymes containing theHis-Glu-Xaa-Xaa-His (or HEXXH) motif which has been shown by X-raycrystallography to form part of the site for binding of the metal atom,commonly zinc. In at least one family of metalloproteases, a glutamicacid residue completes the metal-binding site, HEXXH+E. For example, themost well characterized of the metalloproteases, thermolysin, containsthis motif. The three dimensional structure of thermolysin shows that,in the HEXXH motif, the His residues are zinc ligands and the Gluresidue has a catalytic function. (Methods in Enzymology, Vol. 248,Academic Press, Inc., 1994).

[0006] An interesting variation of the HEXXH+E motif can be found in themetalloprotease family, m16, in which this motif is inverted and seen asHXXEH+E. Members of this family include pitrilysin (Methods inEnzymology, Vol. 248, Academic Press, Inc. ,1994, pp. 684-692) andinsulinase or insulysin (Methods in Enzymology, Vol. 248, AcademicPress, Inc., 1994, pp. 211-215).

SUMMARY OF THE INVENTION

[0007] The present invention relates to the discovery of a heretoforeunknown metalloprotease (MP) found in gram positive microorganisms, usesof the MP in industrial applications, and advantageous strainimprovements based on genetically engineering such microorganisms todelete, underexpress or overexpress that MP. The present invention isbased upon the discovery that MP has overall amino acid relatedness toEscherichia coli pitrilysin.

[0008] The present invention is based upon Applicant's discovery thatthe inverted version of the characteristic metalloprotease amino acidmotif HXXEH+E and putative transmembrane domains exist in Bacillussubtilis MP. Applicant's discovery, in addition to providing a new anduseful protease and methods of detecting DNA encoding other suchproteases in a gram positive microorganism, provides several advantageswhich may facilitate optimization and/or modification of strains of grampositive microorganisms, such as Bacillus, for expression of desired,e.g. heterologous, proteins. Such optimizations, as described below indetail, allow the construction of strains having decreased proteolyticdegradation of desired expression products.

[0009] Due to the relatedness of MP to pitrilysin and insulysin, zincmetalloendopeptidases which have been shown to degrade small peptides ofless than 7kd such as glucagon and insulin, it can be concluded that MPis also an endopeptidase and would be expected to behave similarly topitrilysin and insulysin.

[0010] The present invention encompasses the naturally occurring MPencoded by nucleic acid found in a Bacillus species as well as thenucleic acid and amino acid molecules having the sequences disclosed inSEQ ID NOS: 1 and 2. In one embodiment, the gram positive microorganismis a Bacillus. In a further embodiment, the Bacillus is preferablyselected from the group consisting of Bacillus subtilis, Bacillusstearothermophilus, Bacillus licheniformis and Bacillusamyloliquefaciens. The invention further provides for a metalloproteasethat has at least 80%, preferably at least 90%, most preferably 95%homology with the amino acid sequence of SEQ ID NO: 2. The inventionalso provides for a nucleic acid which encodes a metalloprotease thathas at least 80%, preferably at least 90%, most preferably 95% homologywith the nucleotide sequence shown in SEQ ID NO:1.

[0011] In a preferred embodiment, the present invention encompasses thenaturally occurring MP nucleic acid molecule having the sequence foundin Bacillus subtilis 1-168 strain (Bacillus Genetic Stock Center,accession number 1A1, Columbus, Ohio) in the region of about 1757 kbfrom the point of origin. In another preferred embodiment, the Bacillussubtilis MP nucleic acid and amino acid molecules have the sequences asshown in FIGS. 1A-1F (SEQ ID NOS:1 and 2).

[0012] The present invention provides isolated polynucleotide and aminoacid sequences for Bacillus subtilis MP in FIGS. 1A-1F (SEQ ID NOS:1 and2). Due to the degeneracy of the genetic code, the present inventionencompasses any nucleic acid sequence that encodes the Bacillus subtilisMP amino acid sequence. The present invention provides expressionvectors and host cells comprising a nucleic acid encoding a grampositive MP. The present invention also provides methods of making thegram positive MP.

[0013] The present invention encompasses novel amino acid variations ofgram positive MP amino acid sequences disclosed herein that haveproteolytic activity. Naturally occurring gram positive MP as well asproteolytically active amino acid variations or derivatives thereof,have application in the textile industry, in cleaning compositions andin animal feed.

[0014] The present invention also encompasses amino acid variations orderivatives of gram positive MP that do not have the characteristicproteolytic activity as long as the nucleic acid sequences encoding suchvariations or derivatives would have sufficient 5′ and 3′ coding regionsto be capable of being integrated Into a gram positive organism genome.Such variants would have applications in gram positive expressionsystems where it is desirable to delete, mutate, alter or otherwiseincapacitate the naturally occurring metalloprotease in order todiminish or delete its proteolytic activity. Such an expression systemwould have the advantage of allowing for greater yields of recombinantheterologous proteins or polypeptides.

[0015] The present invention provides methods for detecting grampositive microorganism homologues of B. subtilis MP that compriseshybridizing part or all of the nucleic acid encoding B. subtilis MP withnucleic acid derived from gram positive organisms, either of genomic orcDNA origin. Accordingly, the present invention provides a method fordetecting a gram positive microorganism MP, comprising the steps ofhybridizing gram positive microorganism nucleic acid under lowstringency conditions to a probe, wherein the probe comprises part orall of the nucleic acid sequence shown in FIGS. 1A-1F (SEQ ID NO:1); andisolating the gram positive nucleic acid which hybridizes to said probe.

[0016] The production of desired heterologous proteins or polypeptidesin gram positive microorganisms may be hindered by the presence of oneor more proteases which degrade the produced heterologous protein orpolypeptide. One advantage of the present invention is that it providesmethods and expression systems which can be used to prevent thatdegradation, thereby enhancing yields of the desired heterologousprotein or polypeptide.

[0017] Accordingly, the present invention provides a gram positivemicroorganism that can be used as a host cell having a mutation ordeletion of part or all of the gene encoding MP, io which results in theinactivation of the MP proteolytic activity, either alone or incombination with mutations in other proteases, such as apr, npr, epr,mpr, bpf or isp, or other proteases known to those of skill in the art.In one embodiment of the present invention, the gram positivemicroorganism is a member of the genus Bacillus. In a preferredembodiment, the Bacillus is selected from the group consisting of B.subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus,B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B.lautus and B. thuringiensis. In a further preferred embodiment, theBacillus is Bacillus subtilis.

[0018] In another aspect, the gram positive host cell having one or moremetalloprotease deletions or mutations is further genetically engineeredto produce a desired protein. In one embodiment of the presentinvention, the desired protein is heterologous to the gram positive hostcell. In another embodiment, the desired protein is homologous to thehost cell.

[0019] In another embodiment, a host cell is engineered to produce MP.The gram positive microorganism may be normally sporulating ornon-sporulating. In a preferred embodiment, the gram positive host cellis a Bacillus. In another embodiment, the Bacillus is selected from thegroup consisting of B. subtilis, B. licheniformis, B. lentus, B. brevis,B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B.coagulans, B. circulans, B. lautus and B. thuringiensis. In a furtherpreferred embodiment, the Bacillus host cell is Bacillus subtilis.

[0020] In a further aspect of the present invention, gram positive MP isproduced on an industrial fermentation scale in a microbial hostexpression system. In another aspect, isolated and purified recombinantMP is used in compositions intended for use in the textile industry, incleaning compositions, such as detergents, and in animal feeds.Accordingly, the present invention provides a cleaning composition,animal feed and a composition for the treatment of a textile comprisingMP. The metalloprotease, MP, may be used alone or in combination withother enzymes and/or mediators or enhancers.

[0021] As noted, the present invention provides a cleaning compositioncomprising a metalloprotease, MP, comprising the amino acid sequenceshown in SEQ ID NO:2. Also provided are cleaning compositions comprisinga metalloprotease having at least 80%, preferably 90%, more preferably95% homology with the amino acid sequence shown in SEQ ID NO:2 orcomprising a metalloprotease encoded by a gene that hybridizes with thenucleic acid shown in SEQ ID NO:1.

[0022] Further there is provided an animal feed comprising ametalloprotease, MP, comprising the amino acid sequence shown in SEQ IDNO:2. Also provided are animal feeds comprising a metalloprotease havingat least 80%, preferably 90%, more preferably 95% homology with theamino acid sequence shown in SEQ ID NO:2 or comprising a metalloproteaseencoded by a gene that hybridizes with the nucleic acid shown in SEQ IDNO:1.

[0023] Also provided is a composition for the treatment of a textilecomprising a metalloprotease, MP, comprising the amino acid sequenceshown in SEQ ID NO:2. Also provided are compositions for the treatmentof a textile comprising a metalloprotease having at least 80%,preferably 90%, more preferably 95% homology with the amino acidsequence shown in SEQ ID NO:2 or comprising a metalloprotease encoded bya gene that hybridizes with the nucleic acid shown in SEQ ID NO:1.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIGS. 1A-1D show the DNA and amino acid sequence for Bacillussubtilis MP (YmfH) (SEQ ID NO:1).

[0025] FIGS. 2A-2B show an amino acid alignment of Escherchia colipitrilysin and Bacillus subtilis MP (YmfH). The amino acid motif HXXEH+Eis noted in FIGS. 2A-2B.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Definitions

[0027] As used herein, the genus Bacillus Includes all members known tothose of skill in the art, including but not limited to B. subtilis, B.licheniformis, B. lentus, B. brevis, B. stearothermophilus, B.alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B.lautus and B. thuringiensis.

[0028] The present invention relates to a newly characterizedmetalloprotease (MP) from gram positive organisms. In a preferredembodiment, the gram positive organisms is a Bacillus. In anotherpreferred embodiment, the Bacillus is selected from the group consistingof B. subtilis, B. licheniformis, B. lentus, B. brevis, B.stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans,B. circulans, B. lautus and B. thuringiensis.

[0029] In another preferred embodiment, the gram positive organism isBacillus subtilis and MP has the amino acid sequence encoded by thenucleic acid molecule having the sequence that occurs around 1757kilobases from the point of origin of Bacillus subtilis 1-168.

[0030] In another preferred embodiment, MP has the nucleic acid andamino acid sequence as shown in FIGS. 1A-1F (SEQ ID NOS: 1 and 2). Thepresent invention encompasses the use of amino acid variations of theamino acid sequences disclosed in FIGS. 1A-1D (SEQ ID NO: 2) that haveproteolytic activity. Such proteolytic amino acid variants can be usedin the textile industry, animal feed and cleaning compositions. Thepresent invention also encompasses the use of B. subtilis amino acidvariations or derivatives that are not proteolytically active. DNAencoding such variants can be used in methods designed to delete ormutate the naturally occurring host cell MP.

[0031] As used herein, “nucleic acid” refers to a nucleotide orpolynucleotide sequence, and fragments or portions thereof, and to DNAor RNA of genomic or synthetic origin which may be double-stranded orsingle-stranded, whether representing the sense or antisense strand. Asused herein “amino acid” refers to peptide or protein sequences orportions thereof. A “polynucleotide homologue” as used herein refers toa gram positive microorganism polynucleotide that has at least 80%,preferably at least 90% and more preferably at least 95% identity toB.subtilis MP, or which is capable of hybridizing to B.subtilis MP underconditions of high stringency and which encodes an amino acid sequencehaving metalloprotease activity.

[0032] The terms “isolated” or “purified” as used herein refer to anucleic acid or amino acid that is removed from at least one componentwith which it is naturally associated.

[0033] As used herein, the term “heterologous protein” refers to aprotein or polypeptide that does not naturally occur in the chosen grampositive host cell. Examples of heterologous proteins include enzymessuch as hydrolases including proteases, cellulases, carbohydrases suchas amylases, and lipases; isomerases such as racemases, epimerases,tautomerases, or mutases; oxidases, reductases, transferases, kinasesand phophatases. The heterologous gene may encode therapeuticallysignificant proteins or peptides, such as growth factors, cytokines,ligands, receptors and inhibitors, as well as vaccines and antibodies.The gene may encode commercially important industrial proteins orpeptides, such as proteases, carbohydrases such as amylases andglucoamylases, cellulases, oxidases and lipases. The gene of interestmay be a naturally occurring gene, a mutated gene or a synthetic gene.

[0034] The term “homologous protein” refers to a protein or polypeptidenative or naturally occurring in the chosengram positive host cell. Theinvention includes host cells producing the homologous protein viarecombinant DNA technology. The present invention encompasses a grampositive host cell having a deletion or interruption of the nucleic acidencoding the naturally occurring homologous protein, such as a protease,and having nucleic acid encoding the homologous protein re-introduced ina recombinant form. In another embodiment, the host cell produces thehomologous protein.

[0035] As used herein, the term “overexpressing” when referring to theproduction of a protein in a host cell means that the protein isproduced in greater amounts than in its naturally occurring environment.

[0036] As used herein, the phrase “proteolytic activity” refers to aprotein that is able to hydrolyze a peptide bond. Enzymes havingproteolytic activity are described in Enzyme Nomenclature, 1992, editedWebb Academic Press, Inc.

[0037] The unexpected discovery of the metalloprotease MP found intranslated uncharacterised B.subtilis genomic sequences provides a basisfor producing host cells, expression methods and systems which can beused to prevent the degradation of recombinantly produced heterologousproteins.

[0038] Accordingly, in a preferred embodiment, the host cell is a grampositive host cell that has a deletion or mutation in the naturallyoccurring nucleic acid encoding MP said mutation resulting in deletionor inactivation of the production by the host cell of the MP proteolyticgene product. The host cell may additionally be genetically engineeredto produced a desired protein or polypeptide.

[0039] It may also be desired to genetically engineer host cells of anytype to produce a gram positive metalloprotease. Such host cells areused in large scale fermentation to produce large quantities of themetalloprotease which may be isolated or purified and used in cleaningproducts, such as detergents.

[0040] I. Metalloprotease Sequences

[0041] The present invention encompasses the use of MP polynucleotidehomologues encoding gram positive microorganism metalloproteases MPwhich have at least 80%, preferably at least 90%, more preferably atleast 95% identity to B. subtilis MP as long as the homologue encodes aprotein that has proteolytic activity. A preferred MP polynucleotidehomologue has 96% homology to B. subtilis MP.

[0042] Gram positive polynucleotide homologues of B. subtilis MP may beobtained by standard procedures known in the art from, for example,cloned DNA (e.g., a DNA “library”), genomic DNA libraries, by chemicalsynthesis once identified, by cDNA cloning, or by the cloning of genomicDNA, or fragments thereof, purified from a desired cell. (See, forexample, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual,2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.;Glover, D. M. (ed.), 1985, DNA Cloning: A Practical Approach, MRL Press,Ltd., Oxford, U.K. Vol. I, II.) A preferred source is from genomic DNA.

[0043] As will be understood by those of skill in the art, thepolynucleotide sequence and amino acid sequence disclosed in FIGS. 1A-1Fmay reflect inadvertent errors inherent to nucleic acid sequencingtechnology. The present invention encompasses the naturally occurringnucleic acid molecule having the nucleic acid sequence obtained from thegenomic sequence of Bacillus species.

[0044] Nucleic acid encoding Bacillus subtilis MP starts around 1757kilobases counting from the point of origin in the Bacillus subtilisstrain 1-168 (Anagnostopala, 1961, J. Bacteriol., 81:741-746 or BacillusGenomic Stock Center, accession 1A1, Columbus, Ohio). The Bacillussubtilis point of origin has been described in Ogasawara, N. (1995,Microbiology 141:Pt.2 257-59). Bacillus subtilis MP has a length of 415amino acids. Based upon the location of the DNA encoding Bacillussubtilis MP, naturally occurring B. subtilis MP can be obtained bymethods known to those of skill in the art including PCR technology.

[0045] Oligonucleotide sequences or primers of about 10-30 nucleotidesin length can be designed from the polynucleotide sequence disclosed inFIGS. 1A-1F and used in PCR technology to isolate the naturallyoccurring sequence from B. subtilis genomic sequences.

[0046] Another general strategy for the “cloning” of B. subtilis genomicDNA pieces for sequencing uses inverse PCR. A known region is scannedfor a set of appropriate restriction enzyme cleavage sites and inversePCR is performed with a set of DNA primers determined from the outermostDNA sequence. The DNA fragments from the inverse PCR are directly usedas template in the sequencing reaction. The newly derived sequences canbe used to design new oligonucleotides. These new oligonucleotides areused to amplify DNA fragments with genomic DNA as template. The sequencedetermination on both strands of a DNA region is finished by applying aprimer walking strategy on the genomic PCR fragments. The benefit ofmultiple starting points in the primer walking results from the seriesof inverse PCR fragments with different sizes of new “cloned” DNApieces. From the most external DNA sequence, a new round of inverse PCRis started. The whole inverse PCR strategy is based on the sequentialuse of conventional taq polymerase and the use of long range inverse PCRin those cases in which the taq polymerase failed to amplify DNAfragments. Nucleic acid sequencing is performed using standardtechnology. One method for nucleic acid sequencing involves the use of aPerkin-Elmer Applied Biosystems 373 DNA sequencer (Perkin-Elmer, FosterCity, Calif.) according to manufacturers instructions.

[0047] Nucleic acid sequences derived from genomic DNA may containregulatory regions in addition to coding regions. Whatever the source,the isolated MP gene should be molecularly cloned into a suitable vectorfor propagation of the gene.

[0048] In molecular cloning of the gene from genomic DNA, DNA fragmentsare generated, some of which will encode the desired gene. The DNA maybe cleaved at specific sites using various restriction enzymes.Alternatively, one may use DNAse in the presence of manganese tofragment the DNA, or the DNA can be physically sheared, as for example,by sonication. The linear DNA fragments can then be separated accordingto size by standard techniques, including but not limited to, agaroseand polyacrylamide gel electrophoresis and column chromatography.

[0049] Once the DNA fragments are generated, identification of thespecific DNA fragment containing the MP may be accomplished in a numberof ways. For example, a B. subtilis MP gene of the present invention orits specific RNA, or a fragment thereof, such as a probe or primer, maybe isolated and labeled and then used in hybridization assays to detecta gram positive MP gene. (Benton, W. and Davis, R., 1977, Science196:180; Grunstein, M. and Hogness, D., 1975, Proc. Natl. Acad. Sci. USA72:3961). Those DNA fragments sharing substantial sequence similarity tothe probe will hybridize under stringent conditions. Accordingly, thepresent invention provides a method for the detection of gram positiveMP polynucleotide homologues which comprises hybridizing part or all ofa nucleic acid sequence of B. subtilis MP with gram positivemicroorganism nucleic acid of either genomic or cDNA origin.

[0050] Also included within the scope of the present invention is theuse of gram positive microorganism polynucleotide sequences that arecapable of hybridizing to the nucleotide sequence of B. subtilis MPunder conditions of intermediate to maximal stringency. Hybridizationconditions are based on the melting temperature (Tm) of the nucleic acidbinding complex, as taught in Berger and Kimmel (1987, Guide toMolecular Cloning Techniques, Methods in Enzymology, Vol. 152, AcademicPress, San Diego, Calif.) incorporated herein by reference, and confer adefined “stringency” as explained below.

[0051] “Maximum stringency” typically occurs at about Tm-5° C. (5° C.below the Tm of the probe); “high stringency” at about 5° C. to 10° C.below Tm; “intermediate stringency” at about 10° C. to 20° C. below Tm;and “low stringency” at about 20° C. to 25° C. below Tm. As will beunderstood by those of skill in the art, a maximum stringencyhybridization can be used to identify or detect identical polynucleotidesequences while an intermediate or low stringency hybridization can beused to identify or detect polynucleotide sequence homologues.

[0052] The term “hybridization” as used herein shall include “theprocess by which a strand of nucleic acid joins with a complementarystrand through base pairing” (Coombs, J., (1994), Dictionary ofBiotechnology, Stockton Press, New York, N.Y.).

[0053] The process of amplification as carried out in polymerase chainreaction (PCR) technologies is described in Dieffenbach, C W and G SDveksler, (PCR Primer, a Laboratory Manual, Cold Spring Harbor Press,Plainview, N.Y., 1995). A nucleic acid sequence of at least about 10nucleotides and as many as about 60 nucleotides from B. subtilis MP,preferably about 12 to 30 nucleotides, and more preferably about 20-25nucleotides can be used as a probe or PCR primer.

[0054] The B. subtilis MP amino acid sequences (shown in FIGS. 1A-1F)were identified via a BLAST search (Altschul, Stephen, Basic localalignment search tool, J. Mol. Biol., 215:403-410) of Bacillus subtilisgenomic nucleic acid sequences. B. subtilis MP (YmfH) was identified byits overall nucleic acid identity to the metalloprotease, pitrilysinfrom Eschenchia coli, including the presence of the catalytic domainHXXEH+E as shown in FIGS. 2A-2B.

[0055] II. Expression Systems

[0056] The present invention provides host cells, expression methods andsystems for the enhanced production and secretion of desiredheterologous or homologous proteins in gram positive microorganisms. Inone embodiment, a host cell is genetically engineered to have a deletionor mutation in the gene encoding a gram positive MP such that therespective activity is deleted. In another embodiment of the presentinvention, a gram positive microorganism is genetically engineered toproduce and/or overproduce a metalloprotease of the present invention.

Inactivation of a Gram Positive Metalloprotease in a Host Cell

[0057] Producing an expression host cell incapable of producing thenaturally occurring metalloprotease necessitates the replacement and/orinactivation of the naturally occurring gene in the genome of the hostcell. In a preferred embodiment, the mutation is a non-revertingmutation.

[0058] One method for mutating a nucleic acid encoding a gram positivemetalloprotease is to done the nucleic acid or part thereof, modify thenucleic acid by site directed mutagenesis and reintroduce the mutatednucleic acid into the cell on a plasmid. By homologous recombination,the mutated gene can be introduced into the chromosome. In the parenthost cell, the result is that the naturally occurring nucleic acid andthe mutated nucleic acid are located in tandem on the chromosome. Aftera second recombination, the modified sequence is left in the chromosomehaving thereby effectively introduced the mutation into the chromosomalgene for progeny of the parent host cell.

[0059] Another method for inactivating the metalloprotease proteolyticactivity is through deleting the chromosomal gene copy. In a preferredembodiment, the entire gene is deleted, the deletion occurring in suchas way as to make reversion impossible. In another preferred embodiment,a partial deletion is produced, provided that the nucleic acid sequenceleft in the chromosome is too short for homologous recombination with aplasmid encoding the metalloprotease gene. In another preferredembodiment, nucleic acid encoding the catalytic amino acid residues aredeleted.

[0060] Deletion of the naturally occurring gram positive microorganismmetalloprotease can be carried out as follows. A metalloprotease geneincluding its 5′ and 3′ regions is isolated and inserted into a cloningvector. The coding region of the metalloprotease gene is deleted fromthe vector in vitro, leaving behind a sufficient amount of the 5′ and 3′flanking sequences to provide for homologous recombination with thenaturally occurring gene in the parent host cell. The vector is thentransformed into the gram positive host cell. The vector integrates intothe chromosome via homologous recombination in the flanking regions.This method leads to a gram positive strain in which the protease genehas been deleted.

[0061] The vector used in an integration method is preferably a plasmid.A selectable marker may be included to allow for ease of identificationof desired recombinant microorganisms. Additionally, as will beappreciated by one of skill in the art, the vector is preferably onewhich can be selectively integrated into the chromosome. This can beachieved by introducing an inducible origin of replication, for example,a temperature sensitive origin into the plasmid. By growing thetransformants at a temperature to which the origin of replication issensitive, the replication function of the plasmid is inactivated,thereby providing a means for selection of chromosomal integrants.Integrants may be selected for growth at high temperatures in thepresence of the selectable marker, such as an antibiotic. Integrationmechanisms are described in WO 88/06623.

[0062] Integration by the Campbell-type mechanism can take place in the5′ flanking region of the protease gene, resulting in a proteasepositive strain carrying the entire plasmid vector in the chromosome inthe metalloprotease locus. Since illegitimate recombination will givedifferent results, it will be necessary to determine whether thecomplete gene has been deleted, such as through nucleic acid sequencingor restriction maps.

[0063] Another method of inactivating the naturally occurringmetalloprotease gene is to mutagenize the chromosomal gene copy bytransforming a gram positive microorganism with oligonucleotides whichare mutagenic. Alternatively, the chromosomal metalloprotease gene canbe replaced with a mutant gene by homologous recombination.

[0064] The present invention encompasses host cells having additionalprotease deletions or mutations, such as deletion of or mutation(s) inthe genes encoding apr, npr, epr, mpr and others known to those of skillin the art.

[0065] One assay for the detection of mutants involves growing theBacillus host cell on medium containing a protease substrate andmeasuring the appearance or lack thereof, of a zone of clearing or haloaround the colonies. Host cells which have an inactive protease willexhibit little or no halo around the colonies.

[0066] III. Production of Metalloprotease

[0067] For production of metalloprotease in a host cell, an expressionvector comprising at least one copy of nucleic acid encoding a grampositive microorganism MP, and preferably comprising multiple copies, istransformed into the host cell under conditions suitable for expressionof the metalloprotease. In accordance with the present invention,polynucleotides which encode a gram positive microorganism MP, orfragments thereof, or fusion proteins or polynucleotide homologuesequences that encode amino acid variants of B. subtilis MP, may be usedto generate recombinant DNA molecules that direct their expression inhost cells. In a preferred embodiment, the gram positive host cellbelongs to the genus Bacillus. In a further preferred embodiment, thegram positive host cell is B. subtilis.

[0068] As will be understood by those of skill in the art, it may beadvantageous to produce polynucleotide sequences possessingnon-naturally occurring codons. Codons preferred by a particular grampositive host cell (Murray, E. et al., (1989), Nuc. Acids Res.,17:477-508) can be selected, for example, to increase the rate ofexpression or to produce recombinant RNA transcripts having desirableproperties, such as a longer half-life, than transcripts produced from anaturally occurring sequence.

[0069] Altered MP polynudeotide sequences which may be used inaccordance with the invention include deletions, insertions orsubstitutions of different nucleotide residues resulting in apolynucleotide that encodes the same or a functionally equivalent MPhomologue, respectively. As used herein a “deletion” is defined as achange in the nucleotide sequence of the MP resulting in the absence ofone or more amino acid residues.

[0070] As used herein, an “insertion” or “addition” is that change inthe nucleotide sequence which results in the addition of one or moreamino acid residues as compared to the naturally occurring MP.

[0071] As used herein, “substitution” results from the replacement ofone or more nucleotides or amino acids by different nucleotides or aminoacids, respectively. The change(s) in the nucleotides(s) can eitherresult in a change in the amino acid sequence or not.

[0072] The encoded protein may also show deletions, insertions orsubstitutions of amino acid residues which produce a silent change andresult in a functionally equivalent MP variant. Deliberate amino acidsubstitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues as long as the variant retains itsproteolytic ability. For example, negatively charged amino acids includeaspartic acid and glutamic acid; positively charged amino acids includelysine and arginine; and amino acids with uncharged polar head groupshaving similar hydrophilicity values include leucine, isoleucine,valine; glycine, alanine; asparagine, glutamine; serine, threonine,phenylalanine, and tyrosine.

[0073] The MP polynucleotides of the present invention may be engineeredin order to modify the cloning, processing and/or expression of the geneproduct. For example, mutations may be introduced using techniques whichare well known in the art, i.e., site-directed mutagenesis to insert newrestriction sites, to alter glycosylation patterns or to change codonpreference, for example.

[0074] In one embodiment of the present invention, a gram positivemicroorganism MP polynucleotide may be ligated to a heterologoussequence to encode a fusion protein. A fusion protein may also beengineered to contain a cleavage site located between themetalloprotease nucleotide sequence and the heterologous proteinsequence, so that the metalloprotease may be cleaved and purified awayfrom the heterologous moiety.

[0075] IV. Vector Sequences

[0076] Expression vectors used in expressing the metalloproteases of thepresent invention in gram positive microorganisms comprise at least onepromoter associated with MP, which promoter is functional in the hostcell. In one embodiment of the present invention, the promoter is thewild-type promoter for the selected metalloprotease and in anotherembodiment of the present invention, the promoter is heterologous to themetalloprotease, but still functional in the host cell. In one preferredembodiment of the present invention, nucleic acid encoding themetalloprotease is stably integrated into the microorganism genome.

[0077] In a preferred embodiment, the expression vector contains amultiple cloning site cassette which preferably comprises at least onerestriction endonuclease site unique to the vector, to facilitate easeof nucleic acid manipulation. In a preferred embodiment, the vector alsocomprises one or more selectable markers. As used herein, the term“selectable marker” refers to a gene capable of expression in the grampositive host which allows for ease of selection of those hostscontaining the vector. Examples of such selectable markers include butare not limited to antibiotics, such as, erythromycin, actinomycin,chloramphenicol and tetracycline.

[0078] V. Transformation

[0079] A variety of host cells can be used for the production Bacillussubtilis MP or MP homologues including bacterial, fungal, mammalian andinsects cells. General transformation procedures are taught in CurrentProtocols In Molecular Biology, (Vol. 1, edited by Ausubel et al., JohnWiley & Sons, Inc., 1987, Chapter 9) and include calcium phosphatemethods, transformation using DEAE-Dextran and electroporation. Planttransformation methods are taught in Rodriquez (WO 9514099, publishedMay 26, 1995).

[0080] In a preferred embodiment, the host cell is a gram positivemicroorganism and in is another preferred embodiment, the host cell isBacillus. In one embodiment of the present invention, nucleic acidencoding one or more metalloprotease(s) of the present invention isintroduced into a host cell via an expression vector capable ofreplicating within the Bacillus host cell. Suitable replicating plasmidsfor Bacillus are described in Molecular Biological Methods for Bacillus,Ed. Harwood and Cutting, John Wiley & Sons, 1990, hereby expresslyincorporated by reference; see chapter 3 on plasmids. Suitablereplicating plasmids for B. subtilis are listed on page 92.

[0081] In another embodiment, nucleic acid encoding a metalloprotease(s)of the present invention is stably integrated into the microorganismgenome. Preferred host cells are gram positive host cells. Anotherpreferred host is Bacillus. Another preferred host is Bacillus subtilis.Several strategies have been described in the literature for the directcloning of DNA in Bacillus. Plasmid marker rescue transformationinvolves the uptake of a donor plasmid by competent cells carrying apartially homologous resident plasmid (Contente et al., Plasmid,2:555-571 (1979); Haima et al., Mol. Gen. Genet., 223:185-191 (1990);Weinrauch et al., J. Bacteriol., 154(3):1077-1087 (1983); and Weinrauchet al., J. Bacteriol., 169(3):1205-1211 (1987)). The incoming donorplasmid recombines with the homologous region of the resident “helper”plasmid in a process that mimics chromosomal transformation.

[0082] Transformation by protoplast transformation is described for B.subtilis in Chang and Cohen, (1979), Mol. Gen. Genet., 168:111-115; forB. megaterium in Vorobjeva et al., (1980), FEMS Microbiol. Letters,7:261-263; for B. amyloliquefaciens in Smith et al., (1986), Appl. andEnv. Microbiol., 51:634; for B. thuringiensis in Fisher et al., (1981),Arch. Microbiol., 139:213-217; for B. sphaericus in McDonald, (1984), J.Gen. Microbiol, 130:203; and B. larvae in Bakhiet et al., (1985, Appl.Environ. Microbiol. 49:577). Mann et al., (1986, Current Microbiol.,13:131-135) report on transformation of Bacillus protoplasts andHolubova, (1985), Folia Microbiol., 30:97) disclose methods forintroducing DNA into protoplasts using DNA containing liposomes.

[0083] VI. Identification of Transformants

[0084] Whether a host cell has been transformed with a mutated or anaturally occurring gene encoding a gram positive MP, detection of thepresence/absence of marker gene expression can suggest whether the geneof interest is present. However, its expression should be confirmed. Forexample, if the nucleic acid encoding a metalloprotease is insertedwithin a marker gene sequence, recombinant cells containing the insertcan be identified by the absence of marker gene function. Alternatively,a marker gene can be placed in tandem with nucleic acid encoding themetalloprotease under the control of a single promoter. Expression ofthe marker gene in response to induction or selection usually indicatesexpression of the metalloprotease as well.

[0085] Alternatively, host cells which contain the coding sequence for ametalloprotease and express the protein may be identified by a varietyof procedures known to those of skill in the art. These proceduresinclude, but are not limited to, DNA-DNA or DNA-RNA hybridization andprotein bioassay or immunoassay techniques which include membrane-based,solution-based, or chip-based technologies for the detection and/orquantification of the nucleic acid or protein.

[0086] The presence of the metalloprotease polynucleotide sequence canbe detected by DNA-DNA or DNA-RNA hybridization or amplification usingprobes, portions or fragments of B. subtilis MP.

[0087] VII. Assay of Protease Activity

[0088] There are various assays known to those of skill in the art fordetecting and measuring protease activity. There are assays based uponthe release of acid-soluble peptides from casein or hemoglobin measuredas absorbance at 280 nm or colorimetrically using the Folin method(Bergmeyer, et al., 1984, Methods of Enzymatic Analysis, Vol. 5,Peptidases, Proteinases and their Inhibitors, Verlag Chemie, Weinheim).Other assays involve the solubilization of chromogenic substrates (Ward,1983, Proteinases, in Microbial Enzymes and Biotechnology, (W. M.Fogarty, ed.), Applied Science, London, pp. 251-317).

[0089] VIII. Secretion of Recombinant Proteins

[0090] Means for determining the levels of secretion of a heterologousor homologous protein in a gram positive host cell and detectingsecreted proteins include using either polyclonal or monoclonalantibodies specific for the protein. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescentactivated cell sorting (FACS). These and other assays are described,among other places, in Hampton, R. et al., (1990, Serological Methods, aLaboratory Manual, APS Press, St. Paul Minn.) and Maddox, D E et al.,(1983, J. Exp. Med., 158:1211).

[0091] A wide variety of labels and conjugation techniques are known bythose skilled in the art and can be used in various nucleic and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting specific polynucleotide sequences include oligolabeling, nicktranslation, end-labeling or PCR amplification using a labelednucleotide. Alternatively, the nucleotide sequence, or any portion ofit, may be cloned into a vector for the production of an mRNA probe.Such vectors are known in the art, are commercially available, and maybe used to synthesize RNA probes in vitro by addition of an appropriateRNA polymerase such as T7, T3 or SP6 and labeled nucleotides.

[0092] A number of companies such as Phannacia Biotech (PiscatawayN.J.), Promega (Madison Wis.), and US Biochemical Corp. (Cleveland Ohio)supply commercial kits and protocols for these procedures. Suitablereporter molecules or labels include those radionuclides, enzymes,fluorescent, chemiluminescent, or chromogenic agents as well assubstrates, cofactors, inhibitors, magnetic particles and the like.Patents teaching the use of such labels include U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and4,366,241. Also, recombinant immunoglobulins may be produced as shown inU.S. Pat. No. 4,816,567 and incorporated herein by reference.

[0093] IX. Purification of Proteins

[0094] Gram positive host cells transformed with polynucleotidesequences encoding heterologous or homologous protein may be culturedunder conditions suitable for the expression and recovery of the encodedprotein from cell culture. The protein produced by a recombinant grampositive host cell comprising a mutation or deletion of themetalloprotease activity will be secreted into the culture media. Otherrecombinant constructions may join the heterologous or homologouspolynucleotide sequences to a nucleotide sequence encoding a polypeptidedomain which will facilitate purification of soluble proteins (Kroll, DJ. et al., (1993), DNA Cell Biol., 12:441-53).

[0095] Such purification facilitating domains include, but are notlimited to, metal chelating peptides such as histidine-tryptophanmodules that allow purification on immobilized metals (Porath, J.,(1992), Protein Expr. Purif. 3:263-281), protein A domains that allowpurification on immobilized immunoglobulin, and the domain utilized inthe FLAGS extension/affinity purification system (Immunex Corp, SeattleWash.). The inclusion of a cleavable linker sequence such as Factor XAor enterokinase (Invitrogen, San Diego Calif.) between the purificationdomain and the heterologous protein can be used to facilitatepurification.

[0096] X. Uses of the Present Invention

[0097] MP and Genetically Engineered Host Cells

[0098] The present invention provides genetically engineered host cellscomprising mutations, preferably non-revertable mutations, or deletionsin the naturally occurring gene encoding MP such that the proteolyticactivity is diminished or deleted altogether. The host cell may containadditional protease deletions, such as deletions of the mature subtilisnprotease and/or mature neutral protease disclosed in U.S. Pat. No.5,264,366.

[0099] In a preferred embodiment, the host cell is further geneticallyengineered to produce a desired protein or polypeptide. In a preferredembodiment, the host cell is a Bacillus. In a further preferredembodiment, the host cell is a Bacillus subtilis.

[0100] In an alternative embodiment, a host cell is geneticallyengineered to produce a gram positive MP. In a preferred embodiment, thehost cell is grown under large scale fermentation conditions. In anotherpreferred embodiment, the MP is isolated and/or purified and used in thetextile industry, the feed industry and in cleaning compositions such asdetergents.

[0101] As noted, MP can be useful in formulating various cleaningcompositions. A number of known compounds are suitable surfactantsuseful in compositions comprising the MP of the invention. These includenonionic, anionic, cationic, anionic or zwitterionic detergents, asdisclosed in U.S. Pat. No. 4,404,128 and U.S. Pat. No. 4,261,868. Asuitable detergent formulation is that described in Example 7 of U.S.Pat. No. 5,204,015. The art is familiar with the different formulationswhich can be used as cleaning compositions. In addition, MP can be used,for example, in bar or liquid soap applications, dishcare formulations,contact lens cleaning solutions or products, peptide hydrolysis, wastetreatment, textile applications, as fusion-cleavage enzymes in proteinproduction, etc. MP may comprise enhanced performance in a detergentcomposition (as compared to another detergent protease). As used herein,enhanced performance in a detergent is defined as increasing cleaning ofcertain enzyme sensitive stains such as grass or blood, as determined byusual evaluation after a standard wash cycle.

[0102] MP can be formulated into known powdered and liquid detergentshaving pH between 6.5 and 12.0 at levels of about 0.01 to about 5%(preferably 0.1% to 0.5%) by weight. These detergent cleaningcompositions can also include other enzymes such as known proteases,amylases, cellulases, lipases or endoglycosidases, as well as buildersand stabilizers.

[0103] The addition of MP to conventional cleaning compositions does notcreate any special use limitation. In other words, any temperature andpH suitable for the detergent is also suitable for the presentcompositions as long as the pH is within the above range, and thetemperature is below the described MP's denaturing temperature. Inaddition, MP can be used in a cleaning composition without detergents,again either alone or in combination with builders and stabilizers.

[0104] Proteases can be included in animal feed such as part of animalfeed additives as described in, for example, U.S. Pat. Nos. 5,612,055;5,314,692; and 5,147,642.

[0105] One aspect of the invention is a composition for the treatment ofa textile that includes MP. The composition can be used to treat forexample silk or wool as described in publications such as RD 216,034; EP134,267; U.S. Pat. No. 4,533,359; and EP 344,259.

[0106] MP Polynucleotides

[0107] A B. subtlis MP polynucleotide, or any part thereof, provides thebasis for detecting the presence of gram positive microorganism MPpolynucleotide homologues through hybridization techniques and PCRtechnology.

[0108] Accordingly, one aspect of the present invention is to providefor nucleic acid hybridization and PCR probes which can be used todetect polynucleotide sequences, including genomic and cDNA sequences,encoding gram positive MP or portions thereof.

[0109] The manner and method of carrying out the present invention maybe more fully understood by those of skill in the art by reference tothe following examples, which examples are not intended in any manner tolimit the scope of the present invention or of the claims directedthereto

EXAMPLE I

[0110] Preparation of a Genomic Library

[0111] The following example illustrates the preparation of a Bacillusgenomic library.

[0112] Genomic DNA from Bacillus cells is prepared as taught in CurrentProtocols In Molecular Biology, Vol. 1, edited by Ausubel et al., JohnWiley & Sons, Inc.,1987, Chapter 2. 4.1. Generally, Bacillus cells froma saturated liquid culture are lysed and the proteins removed bydigestion with proteinase K. Cell wall debris, polysaccharides, andremaining proteins are removed by selective precipitation with CTAB, andhigh molecular weight genomic DNA is recovered from the resultingsupematant by isopropanol precipitation. If exceptionally clean genomicDNA is desired, an additional step of purifying the Bacillus genomic DNAon a cesium chloride gradient is added.

[0113] After obtaining purified genomic DNA, the DNA is subjected toSau3A digestion. Sau3A recognizes the 4 base pair site GATC andgenerates fragments compatible with several convenient phage lambda andcosmid vectors. The DNA is subjected to partial digestion to increasethe chance of obtaining random fragments.

[0114] The partially digested Bacillus genomic DNA is subjected to sizefractionation on a 1% agarose gel prior to cloning into a vector.Alternatively, size fractionation on a sucrose gradient can be used. Thegenomic DNA obtained from the size fractionation step is purified awayfrom the agarose and ligated into a cloning vector appropriate for usein a host cell and transformed into the host cell.

EXAMPLE II

[0115] Detection of Gram Positive Microorganisms

[0116] The following example describes the detection of gram positivemicroorganism MP.

[0117] DNA derived from a gram positive microorganism is preparedaccording to the methods disclosed in Current Protocols in MolecularBiology, Chap. 2 or 3. The nucleic acid is subjected to hybridizationand/or PCR amplification with a probe or primer derived from MP.

[0118] The nucleic acid probe is labeled by combining 50 pmol of thenucleic acid and 250 mCi of [gamma ³²P] adenosine triphosphate(Amersham, Chicago Ill.) and T4 polynucleotide kinase (DuPont NEN®,Boston Mass.). The labeled probe is purified with Sephadex G-25 superfine resin column (Pharmacia). A portion containing 10⁷ counts perminute of each is used in a typical membrane based hybridizationanalysis of nucleic acid sample of either genomic or cDNA origin.

[0119] The DNA sample which has been subjected to restrictionendonuclease digestion is fractionated on a 0.7 percent agarose gel andtransferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham N.H.). Hybridization is carried out for 16 hours at 40 degrees C.To remove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1×salinesodium citrate and 0.5% sodium dodecyl sulfate. The blots are exposed tofilm for several hours, the film developed and hybridization patternsare compared visually to detect polynucleotide homologues of B. subtilisMP. The homologues are subjected to confirmatory nucleic acidsequencing. Methods for nucleic acid sequencing are well known in theart. Conventional enzymatic methods employ DNA polymerase Klenowfragment, SEQUENASE® (US Biochemical Corp, Cleveland, Ohio) or Taqpolymerase to extend DNA chains from an oligonucleotide primer annealedto the DNA template of interest.

[0120] Various other examples and modifications of the foregoingdescription and examples will be apparent to a person skilled in the artafter reading the disclosure without departing from the spirit and scopeof the invention, and it is intended that all such examples ormodifications be included within the scope of the appended claims. Allpublications and patents referenced herein are hereby incorporated byreference in their entirety.

1 7 1 1245 DNA Bacillus subtilis 1 atgaaaaaaa gtccaacggc caacggccttgatgtttacg ttttgccgaa aaaaggcttc 60 aacaagacat atgcggtctt tacaacaaagtacggctcga tagataaccg gtttgtccct 120 ttaggtaaaa acgagatggt tcacgtgccggacgggattg ctcactttct tgagcacaag 180 ctgtttgaga aagcggacgg agacgtttttcaagatttca gcaaacaggg ggcttctgcc 240 aatgcgttta cgtcatttac aagaacggcttaccttttct caagcacatc aaatgttgaa 300 cgcaatttag agacgcttat cgatttcgtacaggacccat attttactga aaaaacggtt 360 gaaaaggaaa aagggattat cgggcaggagattaatatgt acgacgataa ccctgattgg 420 aggctttact acggggtcat tgagaacatgtacaaagagc atcctgtcag aattgacata 480 gcgggaacag cggaaagcat ttcacatattacaaaagacc ttctttatga atgctatgaa 540 acgttttatc acccgagtaa catgctccttttcattgtcg gccctgtaga tcctgaagcg 600 attatttctc aggtaagaga aaaccaggggaaaaagccgt atactgatca gccggagatc 660 aaacgagaag aagtgaaaga gcaagaagcggttttccgaa aagaaaaaga gatcaaaatg 720 aacgtgcagg gaccgaaatg ccttgttgggctgaaatcca aaaatccgtt taaattaggc 780 aaagagctct taaagcatga actttcaatgaacttattgc ttgaagctct ttttgccaaa 840 agctctgccc agtatgaatc actttatgaaaaaggatata ttgacgaaac gttcagcttt 900 gattttactg ctgaatatgg gttcggttttgcggcgatcg gcggagatac gcctgagcct 960 gatcaattgg ctgaagacat ttcaagcatgcttttgcgcg ccggtgaact gattactgct 1020 gaaaagattg aacttgccag aaagaaaaagatcggcacat tcttaaaagc gctgaattcc 1080 cctgaataca tcgccaatca atttacccgttatgcgttct tggatatgag cctgtttgat 1140 gtcgtaacgg tactcgagca gattaccctcgaggatgtcc agaacgtaat acaagaggaa 1200 atcgctgcag acagactgac tgtctgcaaggttgttccta aatca 1245 2 415 PRT Bacillus subtilis 2 Met Lys Lys Ser ProThr Ala Asn Gly Leu Asp Val Tyr Val Leu Pro 1 5 10 15 Lys Lys Gly PheAsn Lys Thr Tyr Ala Val Phe Thr Thr Lys Tyr Gly 20 25 30 Ser Ile Asp AsnArg Phe Val Pro Leu Gly Lys Asn Glu Met Val His 35 40 45 Val Pro Asp GlyIle Ala His Phe Leu Glu His Lys Leu Phe Glu Lys 50 55 60 Ala Asp Gly AspVal Phe Gln Asp Phe Ser Lys Gln Gly Ala Ser Ala 65 70 75 80 Asn Ala PheThr Ser Phe Thr Arg Thr Ala Tyr Leu Phe Ser Ser Thr 85 90 95 Ser Asn ValGlu Arg Asn Leu Glu Thr Leu Ile Asp Phe Val Gln Asp 100 105 110 Pro TyrPhe Thr Glu Lys Thr Val Glu Lys Glu Lys Gly Ile Ile Gly 115 120 125 GlnGlu Ile Asn Met Tyr Asp Asp Asn Pro Asp Trp Arg Leu Tyr Tyr 130 135 140Gly Val Ile Glu Asn Met Tyr Lys Glu His Pro Val Arg Ile Asp Ile 145 150155 160 Ala Gly Thr Ala Glu Ser Ile Ser His Ile Thr Lys Asp Leu Leu Tyr165 170 175 Glu Cys Tyr Glu Thr Phe Tyr His Pro Ser Asn Met Leu Leu PheIle 180 185 190 Val Gly Pro Val Asp Pro Glu Ala Ile Ile Ser Gln Val ArgGlu Asn 195 200 205 Gln Gly Lys Lys Pro Tyr Thr Asp Gln Pro Glu Ile LysArg Glu Glu 210 215 220 Val Lys Glu Gln Glu Ala Val Phe Arg Lys Glu LysGlu Ile Lys Met 225 230 235 240 Asn Val Gln Gly Pro Lys Cys Leu Val GlyLeu Lys Ser Lys Asn Pro 245 250 255 Phe Lys Leu Gly Lys Glu Leu Leu LysHis Glu Leu Ser Met Asn Leu 260 265 270 Leu Leu Glu Ala Leu Phe Ala LysSer Ser Ala Gln Tyr Glu Ser Leu 275 280 285 Tyr Glu Lys Gly Tyr Ile AspGlu Thr Phe Ser Phe Asp Phe Thr Ala 290 295 300 Glu Tyr Gly Phe Gly PheAla Ala Ile Gly Gly Asp Thr Pro Glu Pro 305 310 315 320 Asp Gln Leu AlaGlu Asp Ile Ser Ser Met Leu Leu Arg Ala Gly Glu 325 330 335 Leu Ile ThrAla Glu Lys Ile Glu Leu Ala Arg Lys Lys Lys Ile Gly 340 345 350 Thr PheLeu Lys Ala Leu Asn Ser Pro Glu Tyr Ile Ala Asn Gln Phe 355 360 365 ThrArg Tyr Ala Phe Leu Asp Met Ser Leu Phe Asp Val Val Thr Val 370 375 380Leu Glu Gln Ile Thr Leu Glu Asp Val Gln Asn Val Ile Gln Glu Glu 385 390395 400 Ile Ala Ala Asp Arg Leu Thr Val Cys Lys Val Val Pro Lys Ser 405410 415 3 165 PRT Escherichia coli 3 Lys Ser Asp Lys Asp Asn Arg Gln TyrGln Ala Ile Arg Leu Asp Asn 1 5 10 15 Gly Met Val Val Leu Leu Val SerAsp Pro Gln Ala Val Lys Ser Leu 20 25 30 Ser Ala Leu Val Val Pro Val GlySer Leu Glu Asp Pro Glu Ala Tyr 35 40 45 Gln Gly Leu Ala His Tyr Leu GluHis Met Ser Leu Met Gly Ser Lys 50 55 60 Lys Tyr Pro Gln Ala Asp Ser LeuAla Glu Tyr Leu Lys Met His Gly 65 70 75 80 Gly Ser His Asn Ala Ser ThrAla Pro Tyr Arg Thr Ala Phe Tyr Leu 85 90 95 Glu Val Glu Asn Asp Ala LeuPro Gly Ala Val Asp Arg Leu Ala Asp 100 105 110 Ala Ile Ala Glu Pro LeuLeu Asp Lys Lys Tyr Ala Glu Arg Glu Arg 115 120 125 Asn Ala Val Asn AlaGlu Leu Thr Met Ala Arg Thr Arg Asp Gly Met 130 135 140 Arg Met Ala GlnVal Ser Ala Glu Thr Ile Asn Pro Ala His Pro Gly 145 150 155 160 Ser LysPhe Ser Gly 165 4 1500 DNA Bacillus subtilis 4 gagattccta tcgaagactttcttgccaat attgagcatg tcacaaaaga ttcagcttga 60 tacgacttat ttcttaaaagggacggaggg tgcatcttga tcaaaccaat cgaatatgaa 120 cagcttcagg agacgctgtatcatgaaaaa aagtccaacg gccaacggcc ttgatgttta 180 cgttttgccg aaaaaaggcttcaacaagac atatgcggtc tttacaacaa agtacggctc 240 gatagataac cggtttgtccctttaggtaa aaacgagatg gttcacgtgc cggacgggat 300 tgctcacttt cttgagcacaagctgtttga gaaagcggac ggagacgttt ttcaagattt 360 cagcaaacag ggggcttctgccaatgcgtt tacgtcattt acaagaacgg cttacctttt 420 ctcaagcaca tcaaatgttgaacgcaattt agagacgctt atcgatttcg tacaggaccc 480 atattttact gaaaaaacggttgaaaagga aaaagggatt atcgggcagg agattaatat 540 gtacgacgat aaccctgattggaggcttta ctacggggtc attgagaaca tgtacaaaga 600 gcatcctgtc agaattgacatagcgggaac agcggaaagc atttcacata ttacaaaaga 660 ccttctttat gaatgctatgaaacgtttta tcacccgagt aacatgctcc ttttcattgt 720 cggccctgta gatcctgaagcgattatttc tcaggtaaga gaaaaccagg ggaaaaagcc 780 gtatactgat cagccggagatcaaacgaga agaagtgaaa gagcaagaag cggttttccg 840 aaaagaaaaa gagatcaaaatgaacgtgca gggaccgaaa tgccttgttg ggctgaaatc 900 caaaaatccg tttaaattaggcaaagagct cttaaagcat gaactttcaa tgaacttatt 960 gcttgaagct ctttttgccaaaagctctgc ccagtatgaa tcactttatg aaaaaggata 1020 tattgacgaa acgttcagctttgattttac tgctgaatat gggttcggtt ttgcggcgat 1080 cggcggagat acgcctgagcctgatcaatt ggctgaagac atttcaagca tgcttttgcg 1140 cgccggtgaa ctgattactgctgaaaagat tgaacttgcc agaaagaaaa agatcggcac 1200 attcttaaaa gcgctgaattcccctgaata catcgccaat caatttaccc gttatgcgtt 1260 cttggatatg agcctgtttgatgtcgtaac ggtactcgag cagattaccc tcgaggatgt 1320 ccagaacgta atacaagaggaaatcgctgc agacagactg actgtctgca aggttgttcc 1380 taaatcataa acaaaacatccctccagtgt gaggggtgtt tttctgcgga aagaaggaaa 1440 gaggatgaac aaaacagcactaatcaccgg agcaagctgc ggcattggca aaagcatcag 1500 5 82 PRT Escherichiacoli 5 His Tyr Leu Glu His Met Ser Leu Met Gly Ser Lys Lys Tyr Pro Gln 15 10 15 Ala Asp Ser Leu Ala Glu Tyr Leu Lys Met His Gly Gly Ser His Asn20 25 30 Ala Ser Thr Ala Pro Tyr Arg Thr Ala Phe Tyr Leu Glu Val Glu Asn35 40 45 Asp Ala Leu Pro Gly Ala Val Asp Arg Leu Ala Asp Ala Ile Ala Glu50 55 60 Pro Leu Leu Asp Lys Lys Tyr Ala Glu Arg Glu Arg Asn Ala Val Asn65 70 75 80 Ala Glu 6 76 PRT Bacillus subtilis 6 His Phe Leu Glu His LysLeu Phe Glu Lys Ala Asp Gly Asp Val Phe 1 5 10 15 Gln Asp Phe Ser LysGln Gly Ala Ser Ala Asn Ala Phe Thr Ser Phe 20 25 30 Thr Arg Thr Ala TyrLeu Phe Ser Ser Thr Ser Asn Val Glu Arg Asn 35 40 45 Leu Glu Thr Leu IleAsp Phe Val Gln Asp Pro Tyr Phe Thr Glu Lys 50 55 60 Thr Val Glu Lys GluLys Gly Ile Ile Gly Gln Glu 65 70 75 7 233 PRT Bacillus subtilis 7 LysSer Pro Thr Ala Asn Gly Leu Asp Val Tyr Val Leu Pro Lys Lys 1 5 10 15Gly Phe Asn Lys Thr Tyr Ala Val Phe Thr Thr Lys Tyr Gly Ser Ile 20 25 30Asp Asn Arg Phe Val Pro Leu Gly Lys Asn Glu Met Val His Val Pro 35 40 45Asp Gly Ile Ala His Phe Leu Glu His Lys Leu Phe Glu Lys Ala Asp 50 55 60Gly Asp Val Phe Gln Asp Phe Ser Lys Gln Gly Ala Ser Ala Asn Ala 65 70 7580 Phe Thr Ser Phe Thr Arg Thr Ala Tyr Leu Phe Ser Ser Thr Ser Asn 85 9095 Val Glu Arg Asn Leu Glu Thr Leu Ile Asp Phe Val Gln Asp Pro Tyr 100105 110 Phe Thr Glu Lys Thr Val Glu Lys Glu Lys Gly Ile Ile Gly Gln Glu115 120 125 Ile Asn Met Tyr Asp Asp Asn Pro Asp Trp Arg Leu Tyr Tyr GlyVal 130 135 140 Ile Glu Asn Met Tyr Lys Glu His Pro Val Arg Ile Asp IleAla Gly 145 150 155 160 Thr Ala Glu Ser Ile Ser His Ile Thr Lys Asp LeuLeu Tyr Glu Cys 165 170 175 Tyr Glu Thr Phe Tyr His Pro Ser Asn Met LeuLeu Phe Ile Val Gly 180 185 190 Pro Val Asp Pro Glu Ala Ile Ile Ser GlnVal Arg Glu Asn Gln Gly 195 200 205 Lys Lys Pro Tyr Thr Asp Gln Pro GluIle Lys Arg Glu Glu Val Lys 210 215 220 Glu Gln Glu Ala Val Phe Arg LysGlu 225 230

1. A gram positive microorganism having a mutation or deletion of partor all of the nucleic acid encoding a metalloprotease, said mutation ordeletion resulting in the inactivation of the metalloproteaseproteolytic activity, wherein the nucleic acid of the metalloproteasecomprises the sequence shown in SEO ID NO:1 prior to mutation ordeletion.
 2. The gram positive microorganism according to claim 1 thatis a member of the family Bacillus.
 3. The microorganism according toclaim 2 wherein the member is selected from the group consisting of B.subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus,B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B.lautus and B. thuringiensis.
 4. The microorganism of claim 3 whereinsaid microorganism comprises nucleic acid encoding a heterologousprotein.
 5. The microorganism of claim 3 wherein said microorganismcomprises nucleic acid encoding a homologous protein.
 6. Themicroorganism of claim 4 wherein said heterologous protein is selectedfrom the group consisting of hormone, enzyme, growth factor andcytokine.
 7. The microorganism of claim 6 wherein said heterologousprotein is an enzyme.
 8. The microorganism of claim 9 wherein saidenzyme is selected from the group consisting of a protease, acarbohydrase, a lipase, an isomerase, an oxidase, a reductase, atransferase, a kinase and a phosphatase.
 9. A cleaning compositioncomprising a metalloprotease comprising the amino acid shown in SEQ IDNO:2.
 10. A cleaning composition comprising a metalloprotease having atleast 80% homology with the amino acid shown in SEQ ID NO:2.
 11. Acleaning composition comprising a metalloprotease encoded by a gene thathybridizes with the nucleic acid shown in SEQ ID NO:1.
 12. The cleaningcomposition of claim 11, wherein the hybridization takes place under lowstringency conditions.
 13. An animal feed comprising a metalloproteasecomprising the amino acid shown in SEQ ID NO:2.
 14. An animal feedcomprising a metalloprotease having at least 80% homology with the aminoacid shown in SEQ ID NO:2.
 15. An animal feed comprising ametalloprotease encoded by a gene that hybridizes with the nucleic acidshown in SEQ ID NO:1.
 16. The animal feed of claim 15, wherein thehybridization takes place under low stringency conditions.
 17. Acomposition for the treatment of a textile comprising a metalloproteasecomprising the amino acid shown in SEQ ID NO:2.
 18. A composition forthe treatment of a textile comprising a metalloprotease having at least80% homology with the amino acid shown in SEQ ID NO:2.
 19. A compositionfor the treatment of a textile comprising a metalloprotease encoded by agene that hybridizes with the nucleic acid shown in SEQ ID NO:1.
 20. Thecomposition for the treatment of a textile of claim 19, wherein thehybridization takes place under low stringency conditions.
 21. Anexpression vector comprising a nucleic acid encoding a gram positivemetalloprotease comprising the nucleotide sequence shown in SEQ ID NO:1.22. An expression vector comprising a nucleic acid having at least 85%homology with nucleic acid shown in SEQ ID NO:1.
 23. A host cellcomprising an expression vector according to claims 21 or
 22. 24. Amethod for detecting a gram positive microorganism metalloprotease,comprising the steps of (a) hybridizing a gram positive microorganismnucleic acid to a probe, wherein the probe comprises part or all of thenucleic acid sequence shown in SEQ ID NO:1; and (b) isolating the grampositive nucleic acid which hybridizes to said probe.
 25. The method ofclaim 24, wherein the hybridization takes place under low stringencyconditions.